Epigenetic marker for the identification of natural killer cells

Abstract

The present invention relates to a method, in particular an in vitro method for identifying natural killer cells of a mammal, which often express the surface proteins CD 16 and/or CD56, comprising analyzing the methylation status of at least one CpG position in the CX3CR1 and/or FGR and/or NKG7 and/or GNLY genes, in particular their upstream regulatory regions, and in particular the promoter and other conserved regions of the genes CX3CR1 and/or FGR and/or NKG7 and/or GNLY, wherein a demethylation of at least one CpG in the analyzed sample to at least 70% is indicative for CD56 expressing NK cells, which might also be CD8+ or CD8−, CD56 dim or bright, CD 16+ or CD 16− NK cells. The methods of the present invention are useful for the detection and quality assurance and control of NK cells. Furthermore, the present invention relates to a kit for performing the above methods as well as respective uses of the inventive methods or kits. The present invention furthermore provides an improved method for analyzing the methylation status of at least one CpG position in the gene CX3CR1 and/or FGR and/or NKG7 and/or GNLY genes that allows for a precise analysis even from sub-optimal quality samples, such as non-freshly obtained blood, tissue or serum samples.

The invention claimed is: 
     
       1. A method for identifying CD56-expressing natural killer cells in a sample derived from a human, wherein said method comprises the steps of:
 a) obtaining a sample comprising immune cells from said human, 
 b) performing a nucleic acid based assay on the cells in the sample to determine the methylation status of at least one region of a GNLY gene, wherein the methylation status of the at least one region is determined by a method comprising amplifying the at least one region using a primer pair of SEQ ID NO: 146 and 147 and bisulfite sequencing, and 
 c) identifying an immune cell in said sample as a CD56-expressing natural killer cell if the CpG positions in said at least one region as amplified are at least 90% demethylated as determined in step b). 
 
     
     
       2. The method according to  claim 1 , wherein determining the methylation status further comprises the use of a method selected from methylation specific enzymatic digests; analysis selected from CpG island methylation, MSP, HeavyMethyl, MethyLight, and Ms-SNu-PE; or other methods relying on a detection of amplified DNA. 
     
     
       3. The method according to  claim 1 , further comprising an analysis of the markers CD56, CD16 and/or CD8. 
     
     
       4. The method according to  claim 1 , wherein the step of identifying cells as the CD56-expressing natural killer cells comprises a distinction of said natural killer cells from all major peripheral blood cell types or non-blood cells. 
     
     
       5. The method according to  claim 1 , further comprising the step of evaluating an immune status of said human based on said natural killer cells as identified. 
     
     
       6. A method for monitoring a level of CD56-expressing natural killer cells in a human, comprising the method according to  claim 1  and the method further comprising d) determining the amount of CD56-expressing natural killer cells identified in the sample and comparing the amount of CD56-expressing natural killer cells in the sample with an earlier sample taken from the same human and/or with a control sample. 
     
     
       7. The method according to  claim 1 , wherein said human suffers from or is likely to suffer from an autoimmune disease, transplant rejection, cancer, allergy and/or any disease directly correlated to NK cells. 
     
     
       8. The method according to  claim 1 , further comprising d) measuring and/or monitoring the amount of said CD56-expressing natural killer cells in response to chemical and/or biological substances that are provided to said human. 
     
     
       9. The method of according to  claim 1 , wherein the methylation status of at least one additional region is determined by amplifying the at least one additional region using a primer pair selected from SEQ ID NOs: 48 and 49 and SEQ ID NO: 148 and 149. 
     
     
       10. The method according to  claim 1 , wherein the immune cells are obtained from spleen, liver, peripheral blood, bone marrow, thymus, lymph node, or lymphatic fluid. 
     
     
       11. The method, according to  claim 1 , wherein said sample is a blood sample.